US008367395 B2 (12) United States Patent (10) Patent No.: US 8,367,395 B2 Bailey et al. (45) Date of Patent: Feb. 5, 2013 (54) PRODUCTION OFSTEROLS IN (58) Field of Classification Search ........................ None OLEAGNOUSYEAST AND FUNG See application file for complete search history. (75) Inventors: Richard B. Bailey, South Natick, MA (56) References Cited (US); Joshua Trueheart, Concord, MA (US); Kevin T. Madden, Arlington, MA U.S. PATENT DOCUMENTS (US) 2006, OO88903 A1 4/2006 Lang et al. 2009/0324800 Al 12/2009 Bailey et al. (73) Assignee: DSM IP Assets B.V., Heerlen (NL) FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this EP O 486 290 B1 5, 1992 patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 123 days. Merkulov et al. Yeast. Feb. 2000:16(3):197-206.* Chica et al. Curr Opin Biotechnol. Aug. 2005:16(4):378-84.* (21) Appl. No.: 12/443,350 Sen et al. Appl Biochem Biotechnol. Dec. 2007: 143(3):212-23.* 1-1. Veen et al. FEMSYeast Res. Oct. 2003;4(1):87-95.* (22) PCT Filed: Sep. 28, 2007 Madzak et al. J. Biotechnol. Apr. 8, 2004:109(1-2): 63-81.* Athenstaedt et al., “Lipid particle composition of the yeast Yarrowia (86). PCT No.: PCT/US2007/021091 lipolytica depends on the carbon source'. Proteomica, 6:1450-1459 (2006). S371 (c)(1), Merkulov et al., “Cloning and characterization of the Yarrowia (2), (4) Date: May 28, 2009 lipolytica squalene synthase (SQS1) gene and functional complementation of the Saccharomyces cerevisiae erg9 mutation'. (87) PCT Pub. No.: WO2008/130372 Yeast, 16:197-206 (2000). International Search Report for PCT/US2007/021091, mailed Jan. PCT Pub. Date: Oct. 30, 2008 13, 2009. International Preliminary Report on Patentability for PCT/US2007. (65) Prior Publication Data 021091, mailed Mar. 31, 2009. US 2010/0305341 A1 Dec. 2, 2010 * cited by examiner O O Primary Examiner — Christian Fronda Related U.S. Application Data (74) Attorney, Agent, or Firm — Choate, Hall & Stewart, (60) Provisional application No. 60/848,582, filed on Sep. LLP 28, 2006. (57) ABSTRACT (51) Int. Cl. The present invention provides methods for production of one CI2N 9/00 (2006.01) or more sterol compounds. Further provided are methods and CI2N 9/10 (2006.01) systems for producing engineered oleaginous yeast or fungi CI2N I/00 (2006.01) that are capable of production of one or more sterol com CI2N IS/00 (2006.01) pounds, and compositions which utilize the produced Sterol C7H 2L/04 (2006.01) compound(s). (52) U.S. Cl. ................... 435/254.11: 435/183; 435/193; 435/320.1; 536/23.2 9 Claims, 37 Drawing Sheets U.S. Patent Feb. 5, 2013 Sheet 2 of 37 US 8,367,395 B2 U.S. Patent Feb. 5, 2013 Sheet 4 of 37 US 8,367,395 B2 Non-mevalonate Mevalonate Pathway O O Pathway os-coo". Crop He3 g sco 1 2 OH Acetyl-CoA dxS CO2 FIG.3 . COA-SH OH O' O O s OP H3C 1-sca OH 10 3. Acetyl-CoA 2 NADPH +2H COASH dXr (ispC) 2 NADP 9 'oho SOH or 11Š-Ns. S-CoA OP 2 NADPH + 2H 4 CTP O OH COA-SH ispD (ygbp) PP NH2 S 12 OH OH O O N O S o-P-O-P-o N-so ATP OH OH O ADP O O- O OH - ATP Š- OPP ispE (ychB) S. ADP O 13 ATP O W-O NH2 ADP 9 y Q O O "OH r-i-o-, -o N-so S OH OH p O le. O 1 14- SOPPATP ADP ispF (ygbB) -CMP OP O ul-s-s W-O 15 OPP or \o sai-O1 V CO2 OH OH O P 7 1- OPP ispG (gCpE) 16 idi 's- OPP ispH (lytB) OH -s-s OPP 1 7 U.S. Patent Feb. 5, 2013 Sheet 5 Of 37 US 8,367,395 B2 FIG.4 FPP YEAST ERG ERG 9 trans, trans-farensyl diphosphate = presqualene - PP C-14 demethylase ERG 9 squalene squalene monooxygenase ERG 1 2,3-epoxysqualene 2, 3-oxidosqualene lanosterol synthase ERG 7 lanosterol C-14 demethylase t ERG 11 14-demethyllanosterol (4, 4-dimethyl-cholesta-8, 14,24-trienol) C-14 reductase ERG 24 4, 4-dimethylzymosterol (4, 4-dimethyl-cholesta-8,24-dienol) C-4 methyloxidase ERG 25 -a- Vitamin D3 C-4 decarboxylase ERG 26 Zymosterone 3-ketOreductase ERG 27 zymosterol (cholesta-8,24-dienol) C-24 methyltransferase ERG6 fecOStero (ergosta-8,24-dienol) A8-7 isomerase t ERG 2 episterol (ergosta, 7,24-dienol) C-5 desaturase ERG 3 ergosta-5, 7,24-treinol C-22 desaturase ERG 5 ergosta-5, 7,22,24-tetraenol C-24 reductase ERG 4 ergosterol -- vitamin D2 (ergosta-5, 7,22-trienol) U.S. Patent Feb. 5, 2013 Sheet 6 of 37 US 8,367,395 B2 FIG.5 mevatonate pathway Non-mevalonate pathway (Operates in humans) (Operates in some human pathogens/not in humans) isopentenyl diphosphate - I - Dimethylally diphosphate (IPPC) Somerase (DMAPP; C5) Head-to-tail Condensation GPP synthase Phylosierols Geranyl diphosphate -e- Monoterpenes (GPP; C10) FPP synthase (+ IPP) Squalene Synthase Squalene -s- Farnasyl diphosphate -- Sesquiterpenes, (FPP; C15) Triterpenes GGPP synthase Cholesterol, Bile acids (+ IPP) Steroids hormones (in Human) Geranylgeranyl diphosphate -ie- Diterpenes, Carotenoids, (GGPP; C20) Ubiquinones, Menaquinones, Plastoquinones Prenyl diphosphate synthase (+IPP) Polyprenyl diphosphate V Polyprenyl-phosphate (Pol-P) (Pol-PP) P (e.g. C5-C5 in Mycobacteria) U.S. Patent Feb. 5, 2013 Sheet 7 Of 37 US 8,367,395 B2 FIG.6 Hydroxybenzoate soprenyl Biosynthesis Biosynthesis Acetyl-CoA Acetoacetyl-CoA HMG-COA -- HMG-CoA reducase Mevatonate Tyrosine (or phenylalanine) Mevalonate-P 4OH-Phenylpyruvate Mevatonate-PP 4OH-Phenylaciate isopentenyl-PP oil- cle Protein isoprenylation t 4OH-Benzoate Decaprenyl-PP - Farnesyl-PP Z-r Galley | | - -- cis-Prenyltransferase Decaprenoyl trans Squalene Squalene 40H-benzoatetransferase Prenvlrenyltransferase transf SVnthetasey Polyprenyl-PP Decaprenyl-4OH-benzoate Cholesterol / \ Dolichol Dolichy-PP Ubiquinone Carotenoid Cholesterol Biosynthesis Ubiquinone Biosynthesis Biosynthesis U.S. Patent US 8,367,395 B2 U.S. Patent Feb. 5, 2013 Sheet 9 of 37 US 8,367,395 B2 - - - - -nm - r real are C - N - u mi " in to Wu- - -- Y- a-- Y--> -- war U.S. Patent Feb. 5, 2013 Sheet 10 Of 37 US 8,367,395 B2 **** U.S. Patent Feb. 5, 2013 Sheet 11 of 37 US 8,367,395 B2 U.S. Patent US 8,367,395 B2 J. --------> | TÈ era yar ar, wrw Yra r Y ea as -sa -s Mw ars, ex. as M. Yas - N is war Co. i - st d is are tner - - C, CS, “s at s' CSY or --, IN CO O D C co. cc co do is rs is sy wa wala xa wa Y-M We wer ser a weat U.S. Patent US 8,367,395 B2 GE U.S. Patent US 8,367,395 B2 "501-1// U.S. Patent Feb. 5, 2013 Sheet 17 Of 37 US 8,367,395 B2 61.870€HOITWA 6Z8990-IOITWA 6G).OVOJOTVÅ W1670GTdX 99Z99909||7030GTdX 81879903| U.S. Patent US 8,367,395 B2 U.S. Patent Feb. 5, 2013 Sheet 20 Of 37 US 8,367,395 B2 FIG.9A Schematic diagram of certain plasmids used herein pMB4591 6680 bp kan R hmg1-trunc Ban HI Acc65. Kpni Mll I Spel ACC65 Kpni Sna BI U.S. Patent Feb. 5, 2013 Sheet 21 Of 37 US 8,367,395 B2 FIG.9B Schematic diagram of certain plasmids used herein Accó51 Kpni ampr carb(i-) pMB4660 : 8046 bp exon3 ill Mlul kan R &\ RN / Spe Xho Aw Ni (8261) HindIII (360) - ERG9Orf pMB4789 pcrmut (TioA)(1890)s KAN erg9::URA3 -- BstEIII (2231.) Spe I (2290) Alw NI (5935) F1 Ori --- AfLII (3267) URA3Orf Apa I (5220) Sma I (3695) Aw NI (5134) N. Nsi I (4204) ERG9 3'flank U.S. Patent Feb. 5, 2013 Sheet 22 Of 37 US 8,367,395 B2 FIG.9C Schematic diagram of certain plasmids used herein Acc65 (250) KpnI (254) w Spel (453) NheI (658) NotI (665) Mlul (672) Spel (810) AMP Sal I (1166) if ura3 (1549) KANr Xbal (2734) F1 Or. Spel (8353) Mlul (1) / Spel (139) carrP(-) Spel (6349) Kpnl (6150) i. XbalNot I (2303)(232 I) AccG5I (6146) U.S. Patent Feb. 5, 2013 Sheet 23 Of 37 US 8,367,395 B2 FIG.9D pUC ori ampR Kan R pMB4628 8470 bp exOn2 pMB4638 9596 bp exOn3 U.S. Patent Feb. 5, 2013 Sheet 24 of 37 US 8,367,395 B2 FIG.9F Kan R ampR pMB4692 Kan R U.S. Patent Feb. 5, 2013 Sheet 25 Of 37 US 8,367,395 B2 FIG.9H pUC ori pMB4698 8653 bp ACC65 (250 FIG.91 / ci. Ea Spel (453) HMG1 trune AMPrs BamHI (1783) AccG5 (1883) Kpni (1887) MIul (2178) KANr Spel (2316) Xba I (4240) URA3Of U.S. Patent Feb. 5, 2013 Sheet 26 of 37 US 8,367,395 B2 FIG.9/ pUC ori AMP Acc65 (1008) Kpnl (1012) Spel (1211) KANr. Ay 4- Nhe I (1453) CrtW (Parva.) Mlu I (2220) F1 Ori A Spel (2358) N. AccG5 (2588) Xba I (5832) - Kpnl (2592) Not I (5814) S-Accesi (4030) ADE 1 of Kpnl (4034) FIG.9K pUC ori AMPr AccG5I (1008) Kpnl (1012) Spel (1211) KANr crtW (Aurant.) i-Miul (2377) fis Spel (2515) - Acc65 (2745) Xbal (5989) Kpnl (2749) Nott (5971) AccG5 (4187) ADE 1 of Kpnl (41.91) U.S. Patent Feb. 5, 2013 Sheet 27 Of 37 US 8,367,395 B2 FIG.9L AccG5I (241), Kpn I (245) Spel (444) Nhe I (650) Not I (657) Miu I (664) AMP Bam HI (2028) Spel (2034) Sal I (2398) URA3 Acc 65I (250) Kpn I (254) Spel (453) Mlul (882) CrtZ (Xanth.) Mlul (1178) AMP Spel (1334) Sal I (1690) KANr URA3Orf U.S.